Effects of glycoprotein and basement membrane synthesis inhibitors on the growth of cultured renal collecting duct epithelium

Summary The biochemical and morphological extent of glycoprotein synthesis inhibition of cellular and extracellular proteins was studied on cultured renal collecting duct (CD) epithelium. We found that tunicamycin (4 g/ml) inhibits the glycosylation of a 150,000 d glycoprotein (gp_CDI). A 85,000 d glycoprotein (gp_CDII) was not affected. The inhibition by tunicamycin demonstrates that gp_CDI has characteristics of a N-glycan, whereas gp_CDII seems to be an O-glycan. 6-diazo-5-oxo-norleucine (4×10^–5M) which was used as glutamine analogue, did not show a comparable inhibitory effect as seen with tunicamycin. The lack of effect of norleucine demonstrates that glutamine is not the locus of glycosylation in both proteins. However, because of the tunicamycin inhibition it points to asparagine as the site of glycosylation in the gp_CDI. Long term cultures of the tissue up to 15 days in the presence of tunicamycin and norleucine and of substances usually used as basement membrane inhibitors, such as hydroxy-d-proline (1 mM), l-azetidine-2-carboxylic acid (1 mM) and o- and p-nitrophenyl-xylopyranoside (1 mM), revealed that it is possible to eliminate completely the fibroblasts beneath the cultured epithelium and within the degenerating corematerial. Experiments with hydroxy-d-proline showed the most striking effect. Experiments with l-azetidine-2-carboxylic acid and nitrophenyl-xylopyranoside resulted in the elimination of fibroblasts and dedifferentiation of the collecting duct epithelium.